The invention relates both to the field of genetic manipulation by means of the recombinant DNA technology for the production of certain proteins and/or particles that consist of one or more of these proteins, and to the fields of diagnostics and vaccine preparation. The invention concerns certain viral proteins, which may or may not be in the form of virus-like particles, which proteins or particles can for instance be used in assays for detecting antibodies directed against these proteins, or can be used to obtain such antibodies, or can be used to accomplish protection against the virus, or can be used for the incorporation therein of epitopes of proteins of other pathogenes to accomplish protection against these other pathogenes (and thus offers various possibilities of use for vaccination purposes).
More particularly, the invention relates to the coat proteins VP1 and VP2 of the human parvovirus B19 and to virus like particles that consist of VP2 or of VP1 and VP2. The invention further comprises genetic information in the form of recombinant expression vectors which contain the genes coding for these proteins, and organisms that have acquired the ability to produce the proteins and/or particles in question owing to genetic manipulation using such vectors.
The human parvovirus B19 was serendipitously discovered in 1975 in serum samples of some healthy blood donors. Since that time it has been found that the virus causes erythema infectiosium 13 also known as xe2x80x9cfifth diseasexe2x80x9dxe2x80x94and of the so-called xe2x80x9caplastic crisisxe2x80x9d in patients with chronic hemolytic anemia. The B19 virus is further associated with abortion and fetal death, with arthritis and with chronic anemia in immuno-deficient patients. Infections may also occur under other syndromes or occur entirely asymptomatically.
Infections with this virus, which is found throughout the world, usually occur in epidemics which take place about every 3-6 years, but may occur sporadically in intervening years. Today, fourteen years after the discovery of the B19 virus, the diagnostics for infection with the virus are still performed in only a limited number of laboratories in the world. Because the virus cannot be demonstrated anymore in the patients at the time when the symptoms arise (viremia and virus excretion precede the symptoms), diagnostics must focus on demonstrating B19-specific (IgMr)-antibodies.
To this end (and also for the preparation of suitable vaccines, for example) it is necessary to have a sufficient supply of B19-antigen for setting up the tests. What is lacking, however, is a suitable in vitro cell culture system for propagating the virus, with which sufficient antigen can be obtained.
The existing parvovirus B19 diagnostics are performed with virus antigen which becomes available more or less by chance (screening blood donors offers an estimated chance of 1 in 50,000 that viremic blood is found).
For these reasons there is a great need for antigen which is produced using recombinant DNA techniques. Accordingly, various proposals in this direction have already been made, but none of them has proved really useful for the construction of a diagnostic test.
The present invention is based on the use of an expression vector system that was developed fairly recently, viz. the xe2x80x9cBaculovirus Expression Vector Systemxe2x80x9d. In this system use is made of a recombinant virus vector of the baculo-virus Autoaraphica californica nuclear polyhedrosis virus (AcNPV) to express the B19 virus proteins in insect cells: Spodoptera frugiperda (Sf9). This system offers many advantages over the current systems of expression vectors:
a) In view of the use for diagnostic and possibly therapeutic (vaccination) purposes, no cross reactivity is to be expected against proteins of the baculovirus or the insect cells (in proteins which are expressed in E.coli, this cannot always be precluded).
b) The virus proteins can be produced in large amounts (1-500 mg/1) up to even 50-75% of the total protein, detected on SDS-polyacrylamide gel (Summers and Smith, 1986, a manual of methods for baculovirus vector and insect cell culture procedures; Yong Kang, 1988; Adv. in Virus Res. 35, 177-192). These are considerably larger amounts than those produced in prokaryotic expression systems or in Chinese hamster ovary cells, as described by Kajigaya et al. (Blood 75(5), suppl.1, 44a, abstr. 86; 1988).
c) The proteins can be produced as non-fusion proteins, in contrast to for instance the B19 protein, which has been produced as a fusion protein in E.coli by Sisk and Berman (Biotechnology 5, 1077-1080, 1987). This recombinant xcex2-galactosidase-B19 fusion protein goes into solution only in the presence of sodium dodecylsulphate (SDS). The proteins VP1 and VP2 expressed in insect cells in accordance with the invention, by contrast, can easily be dissolved by sonification of the cells in a buffer which contains 25 mM NaHCO3 and 20 mg/l NaN3 (pH 9.5). In such a treatment 95% of the cellular proteins go over into the soluble supernatant fraction.
d) The proteins can be produced in an insect cell line which is easy to culture, as opposed to the production of virus proteins in human erythroid bone marrow cells (Ozawa et al., 1987; Blood 70, 384-391) or human foetal erythroid liver cells (Yaegashi et al., 1989; J. Virol. 63, 2422-2426). 
e) Because in the baculovirus expression vector system pre- and post-translation modifications occur, such as phosphorylation, glycosylation, signal peptide split-off and the removal of introns by splicing, the system is potentially very suitable for the production of biologically active proteins with a (virtually) native structure (Yong Kang, 1988; Adv. in Virus Res. 35, 177-192). In this system VP1 and VP2 of B19 can be expressed both separately and collectively. Moreover, the possibility exists that virus-like particles are spontaneously formed from one or more of these proteins.
f) An additional advantage of the baculovirus is that it does not multiply in mammalian cells and hence is not pathogenic for humans, which makes it much safer to work with and utilize this system.
According to the invention it has actually been accomplished to produce in a high yield the coat proteins VP1 and VP2 of the human parvovirus B19 in an antigenically active form as non-fusion proteins, as virus-like particles or not, using the baculovirus expression system in insect cells (Spodoptera frugiperda). Further, it has been accomplished to develop, using the B19 virus proteins producing insect cells, a specific and sensitive immunofluorescence-assay (IFA) and a specific and sensitive Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) for the detection of antibodies directed against the B19 virus proteins. However, on the basis of the B19 virus proteins and virus-like particles produced in insect cells in conformity with the invention, other diagnostic assays can be developed as well, such as a Radio-Immuno-Assay (RIA) or an agglutination test.
The invention is primarily embodied in recombinant VP1 and VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of the B19 virus protein VP1 and/or VP2. The invention further comprises recombinant virus-like particles which consist of VP2 protein or of VP1 and VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of VP2 protein or of VP1 and VP2 protein.
Further, the invention is embodied in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information which is necessary for expression of VP1 and/or VP2 protein of the human parvovirus B19.
The invention further provides a method of producing VP1 and/or VP2 protein of the human parvovirus B19 (optionally in the-form of virus-like particles which are composed of VP2 protein or of both proteins) by culturing Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information which is necessary for expression of the B19 virus protein or the B19 virus proteins. Optionally and preferably the B19 virus protein formed in the cells and/or the virus-like particles formed in the cells and consisting of VP2 protein or of VP1 and VP2 protein are isolated from the cells. A suitable method for that purpose comprises a sonification of the cells in a buffer which contains 25 mM NaHCO3 and 20 mg/l NaN3 (pH 9.5). The result of such a treatment is that a great part of the proteins present in the cells, for instance 95%, are obtained in dissolved form in the supernatant. By known per se purification methods, the B19 virus proteins can be isolated at a higher purity.
The invention is also embodied in recombinant baculovirus expression vectors, equipped with the genetic information which is necessary for expression of VP1 and/or VP2 protein of the human parvovirus B19 in Spodoptera frugiperda cells. Preferred embodiments of such recombinant baculovirus expression vectors are the plasmids pAcB19VP1-YM1 and pAcB19VP2-YM1, to be described hereinafter.
The invention is further embodied in recombinant baculoviruses, equipped with the genetic information which is necessary for expression of VP1 and/or VP2 protein of the human parvovirus B19 in Spodoptera frugiperda cells. Preferred embodiments of such recombinant baculoviruses are the viruses AcB19VP1L and AcB19VP2L, to be described hereinafter.
The invention further comprises the use of recombinant VP1 and/or VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of the B19 virus protein, in an assay for detecting antibodies directed against the B19 virus protein in a sample to be tested. The invention comprises the use of recombinant virus-like particles which consist of VP2 protein or of VP1 and VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of these B19 virus proteins, in an assay for detecting antibodies directed against the B19 virus in a sample to be tested. In preferred embodiments of the invention, this concerns the use of Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information which is necessary for expression of VP1 and/or VP2 protein of the human parvovirus B19, in an assay for detecting antibodies directed against the B19 virus protein in a sample to be tested, more particularly in an IFA or ELISA for detecting antibodies directed against the B19 virus protein in a sample to be tested.
The invention also comprises a vaccine preparation for inducing an immune response which provides protection against the human parvovirus B19, comprising recombinant VP1 and/or VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of the B19 virus protein, or an antigenically active portion of this recombinant B19 virus protein, in combination with one or more carriers and/or adjuvants suitable for vaccination purposes, and further, a vaccine preparation for inducing an immune response which provides protection against the human parvovirus B19, comprising recombinant virus-like particles which consist of VP2 protein or of VP1 and VP2 protein of the human parvovirus B19, formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of these B19 virus proteins, in combination with one or more carriers and/or adjuvants suitable for vaccination purposes.
The invention further comprises the use of recombinant VP1 and/or VP2 protein of the human parvovirus B19 (or virus-like particles consisting of VP2 or of VP1 and VP2), formed in Spodoptera frugiperda cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of the B19 virus protein, or with an antigenically active portion of this recombinant B19 virus protein, for inducing an immune response which provides protection against the human parvovirus B19.
The invention also comprises the use of virus-like particles consisting of VP2 protein or VP1 and VP2 protein of the human parvovirus B19, into which one or more epitopes of proteins of other pathogens have been incorporated, for inducing an immune response which provides protection against these other pathogens.
In the experimental section to follow hereinbelow, it is shown by way of explanation and illustration how the invention was carried out and can be carried out. As shown by the Examples, the DNA sequences coding for the structural proteins VP1 and VP2 of the human parvovirus B19 were isolated from the B19 virus from the serum of a patient. Then, via subcloning steps in pUC19 and pUC7, the B19-DNA was cloned into the baculovirus vector pAcYM1 behind the promoter for the polyhedrin gene of the baculovirus. By means of cotransfection of this recombinant vector with wild type baculovirus DNA, followed by recombination in the insect cells (Spodoptera frugiperda), finally, recombinant virus was isolated which, after infection in the insect cells, led to the production of the coat proteins VP1 and VP2 of B19, which were or were not in the form of virus-like particles. Using these B19 proteins, sensitive and specific IFA and ELISA tests were developed, enabling fast and simple detection of B19-specific antibodies. The proteins produced in this manner, which may or may not be in the form of virus-like particles, may also serve as easily obtainable antigens for other diagnostic tests, such as RIA""s and agglutination tests and for the (possible) production of vaccines and subunit vaccines.